Increased serum caspase-1 in adult-onset Still’s disease

Background Caspase-1 is a crucial component in the inflammasome activation cascade. This study evaluated the potential of serum caspase-1 level as an inflammatory biomarker in patients with adult-onset Still’s disease (AOSD). Methods The study included 51 consecutive patients diagnosed with AOSD based on the Yamaguchi criteria, 66 patients with rheumatoid arthritis (RA) as disease control, and 36 healthy controls (HCs). Serum caspase-1 concentrations were measured using enzyme-linked immunosorbent assay. The serum 69 cytokine levels were analyzed using a multisuspension cytokine array in patients with AOSD, and a cluster analysis of each cytokine was performed to determine specific molecular networks. Results Patients with AOSD had significantly increased serum caspase-1 levels versus patients with RA (p < 0.001) and HCs (p < 0.001). Additionally, serum caspase-1 demonstrated significant positive correlations with AOSD disease activity score (Pouchot score, r = 0.59, p < 0.001) and serum ferritin (r = 0.54, p < 0.001). Furthermore, among patients with AOSD, significant correlations existed between serum caspase-1 and inflammatory cytokines, including interleukin-18. Immunoblot analysis detected the cleaved form of caspase-1 (p20) in the serum of untreated patients with AOSD, not in those from patients with inactive AOSD receiving immunosuppressive treatments. Conclusions Caspase-1 is a useful biomarker for AOSD diagnosis and monitoring. Caspase-1 activation could be correlated with the inflammatory component of AOSD, specifically through proinflammatory cytokine induction via inflammasome activation cascades.

Caspase-1 is the prime member of the inflammatory caspases, and it activates pro-IL-1β or pro-IL-18 [6].Activated caspase-1 may result in the processing of IL-18 and IL-1β into mature forms, which play an important role in cytokine cascades in AOSD, during the inflammasome activation processes [7].Previous studies revealed that serum caspase-1 was elevated in patients with inflammatory disorders, including neurological or hepatic diseases [8,9].However, the use of caspase-1 as a serum biomarker for autoinflammatory disorders remains unknown, and the role of caspase-1 in rheumatic diseases is still unclear.This study investigated the clinical relevance of caspase-1 as an autoinflammatory biomarker in AOSD, considering the importance of inflammasomes in its pathogenesis.We analyzed serum caspase-1 levels in patients with AOSD versus those with rheumatic arthritis (RA) and healthy controls (HCs).Additionally, we investigated the clinical correlations of caspase-1 with inflammatory cytokines and disease parameters in patients with AOSD.

Patients and study design
This study enrolled 51 untreated patients with AOSD who were treated at Fukushima Medical University Hospital Department of Rheumatology from January 1995 to April 2020.All patients included had to be �17 years old to be diagnosed with AOSD following Yamaguchi's diagnostic criteria [10] after excluding those with infectious, neoplastic, and autoimmune disorders.
We additionally collected serum samples from 18 patients with AOSD treated during remission to investigate the longitudinal changes.As controls, 36 HCs (12 men and 24 women, median age: 39 years, interquartile range [IQR]: 32-45 years) were included.HCs lacked chronic medical diseases or conditions and did not take prescription medications or over-the-counter medications within 7 days.Additional independent sets consisting of 66 patients with rheumatoid arthritis (RA) were used to identify the specificity of the values of caspase-1 in patients with AOSD.Patients diagnosed with RA in Fukushima Medical University Hospital were randomly selected.This study was conducted following the principles of the Declaration of Helsinki and approved by the institutional review boards of Fukushima Medical University (No. 2021-290; approval date: April 16, 2023) and the National Center for Global

Immunoblot analysis
Human serum samples were diluted 10-fold with PBS.We separated this diluted human serum sample (total serum amounts: 2 μl) plus protein loading buffer of 5 μl by NuPAGE 3%-8% Tris-acetate gel electrophoresis (Invitrogen, Carlsbad, CA, USA).Proteins were electrophoretically transferred onto an Invitrogen polyvinylidene fluoride membrane and incubated overnight at 4˚C with a blocking solution [5% non-fat milk in Tris-buffered saline with 0.05% Tween 20 (TTBS)].The blocked membrane was incubated with rabbit anti-human caspase-1 (full-length p48, D7F10, Cell Signaling Technology, Boston, MA, USA) antibody or cleaved caspase-1 antibody (p20 D57A2, Cell Signaling) at 1:1000 dilution with 1% non-fat milk in TTBS for 1 h at room temperature and then washed five times with TTBS buffer for 10 min each time at room temperature with constant shaking.The membrane was then incubated with horseradish peroxidase-conjugated second antibody (1:3000 dilution; Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h at room temperature and washed five times with TTBS buffer for 10 min each time at room temperature with constant shaking.An electrochemiluminescence western blotting kit (Amersham, Little Chalfont, UK) was used for immunodetection analysis.Images of the developed film were scanned using a LAS-3000 image analyzer (Fujifilm, Tokyo, Japan).As an internal control, anti-human transferrin (1:7000 dilution; mouse, 661771-1-Ig, Proteintech), was used to detect transferrin using the above-described process.

Statistical analysis
Data are presented as medians and IQR for continuous variables and as frequencies and percentages for qualitative variables.Spearman's rank correlation test was used to calculate correlations between pairs of serum markers.We performed the multivariate classification algorithm of random forest analysis using the R package RandomForest (http://cran.r-project.org/web/packages/randomForest/) version 4.6.12software, as previously described, to rank the cytokine levels [11].Mann-Whitney's U test was used to compare group differences.Wilcoxon signed-rank tests were used to evaluate changes pre-and post-treatment.R software (version 4.3.1)and Statistical Package for the Social Sciences Statistics software (version 29.0;IBM Corp., Armonk, NY, USA) were used for statistical analyses.The receiver operating characteristic (ROC) curve was used to identify the cut-off value for caspase-1 in distinguishing between AOSD and RA.All reported p-values were two-sided, and a P-value of <0.05 was considered statistically significant.Bonferroni's correction was applied to the multiplex analysis.

Demographic data of patients with AOSD
This study included 51 patients with AOSD (88.2% female; median age: 40 years, IQR: 28-56), whose serum samples were collected in the active state.The laboratory parameters include a complete blood count, liver function tests, C-reactive protein (CRP), and ferritin.Table 1 summarizes the baseline characteristics and laboratory data.The principal clinical symptoms included a high spiking fever (82.4%), skin rash (68.7%), arthralgia (57.8%), sore throat (41.2%), and splenomegaly (39.2%).Patients with AOSD demonstrated elevated median levels of biological markers that represent disease activity, such as CRP (median: 6.8 mg/dl, IQR: 2.9-10.9)and ferritin (median: 1,159 pg/ml, IQR: 310-3,887).The maximum prednisolone dose during the active phase, the number of patients who received steroid pulse, and concomitant immunosuppressive drugs are described.

Serum caspase-1 levels in patients with AOSD
Serum caspase-1 levels were determined using ELISA in patients with AOSD, those with RA, and HCs.Patients with AOSD demonstrated significantly higher caspase-1 levels [median: 419.33 ng/ml, IQR (79.19-555.84)]compared to those with RA (6.98 ng/ml, p < 0.001) and HCs (5.85 ng/ml, p < 0.001) (Fig 1).We analyzed the cut-off value for caspase-1 in distinguishing between AOSD and RA by obtaining the ROC curve, which revealed 26.9 ng/mL (sensitivity of 100.0% and specificity of 93.9%).The area under the ROC curve was 0.987 (Fig 2).
Moreover, we evaluated the correlations of serum caspase-1 with various clinical parameters in patients with AOSD.Serum caspase-1 did not correlate with leucocyte counts or transaminases, but it was significantly correlated with serum ferritin and the Pouchet score (Fig 3), which correspond to the disease activity of AOSD [12].However, no correlation was observed between CRP and caspase-1 (r = 0.143, p = 0.356) (data was not shown in the figure).Additionally, the comparison of caspase-1 between MAS and non-MAS revealed no significant difference (p = 0.189).
We included 18 patients with 2 longitudinal samples (at least 1 month apart) to investigate the longitudinal changes in caspase-1.The longitudinal study followed 8 patients with active AOSD until they became inactive, and then they were resampled, revealing 6 non-MAS and 2 cases were MAS cases.Serum caspase-1 levels significantly decreased (Fig 4) alongside ferritin and Pouchot's score after immunosuppressive treatments.

Detection of circulating cleaved caspase-1 in untreated patients with AOSD
Serum samples from patients with AOSD patients underwent immunoblot analysis using an anti-full-length caspase-1 antibody to detect the circulating caspase-1.Single bands with molecular weights (45 kDa) were detected in the sera from patients with AOSD as well as in those from HCs, indicating that they originated from the heavy chain of serum-containing IgG.However, the full-length caspase-1 band (p48) was not detected in serum from patients with AOSD (Data not shown).Caspase-1 activation occurs during its release, and it is not an intracellular event in response to exogenous inflammasome activation [13].Extracellular caspase-1 is induced during its cleavage step in pro-IL-1β processing [14].Therefore, immunoblot analysis using anti-cleaved caspase-1 antibody (p20

Discussion
Inflammasomes are multiprotein complexes that serve as pattern-recognition receptors in the immune system [15].Inflammasomes can be activated by recruiting and activating caspase-1 upon sensing pathogen-associated molecular patterns [16].Activated caspase-1 converts IL-1β and IL-18 precursors into their mature forms [3], thereby playing an important role in the pathogenesis of autoinflammatory diseases [17].In this study, ELISA analysis revealed significantly higher caspase-1 levels in patients with active AOSD, and the elevated caspase-1 levels were correlated with serum ferritin and the Pouchet score, which are the systemic activation markers for AOSD.Furthermore, serum caspase-1 was correlated with serum IL-18, which is a crucial mediator maturated by inflammasomes and is presumed as an AOSD-specific biomarker.The correlation of caspase-1 with the disease activity scores for AOSD revealed that the elevated caspase-1 levels may reflect the inflammasome activation status, which is implicated in the pathogenesis of AOSD.Altogether, caspase-1 serves as a novel biomarker for assessing and monitoring AOSD disease activity.Inflammasomes are composed of three main components: NLRP3, ASC (an adaptor protein termed as apoptosis-associated speck-like protein containing a caspase recruitment domain), and pro-caspase-1, which mounts an inflammatory response against cellular damage [18].Inflammasome activation, in turn, promotes caspase-1 activation [19].Activated caspase-1 cannot be endogenously detected in the cytosol of innate immune cells upon inflammasome activation [20].However, the cleaved caspase-1 subunit was detected, indicating that mature caspase-1 is released along with processed IL-1β and IL-18 during the inflammasome activation cascade and serves as a marker for inflammasome activation [21].Activated caspase-1 triggers Gasdermin D (GSDMD) and pro-IL−1β or pro-IL-18 cleavage [22].The cleaved GSDMD promotes pore formation in the plasma membrane and further facilitates the release of these inflammatory   mediators [23].hence, we investigated the sera of patients with AOSD for cleaved caspase-1 using immunoblot analysis.We revealed that the cleaved form of caspase-1, p20, was detectable in the serum of patients with AOSD.Active patients with AOSD had markedly increased expression of caspase-1 with its cleaved form.However, the cleaved form of caspase-1 was exclusively found in untreated patients with AOSD, and it was undetectable among patients with AOSD in the remission state receiving immunosuppressive therapy.Pyroptosis, a caspase-1-dependent type of programmed cell death, recently promoted IL-1β and IL-18 secretion during the inflammatory response [24].Caspase-1 has activated the transcription factor NF-kB, independent of its enzymatic activity, which may cause inflammation in vivo [25].This indicates the presence of cross-talk between blood caspase-1 and the inflammatory mediators in the inflammatory disorder.Altogether, caspase-1 may have a potential role in inflammatory cascades by improving inflammatory cytokine production.
Recent studies have confirmed the direct association of IL-1β or IL-18 activation by caspase-1 with inflammatory environments [26].Overexpressed caspase-1 has been demonstrated in the aortas of patients with coronary atherosclerosis [27].Caspase-1 can be widely activated in endothelial cells and circulating innate immune cells in autoinflammation [28].However, the role of caspase-1 in AOSD remains unclear.In our study, upregulated circulating caspase-1 was associated with inflammasome activation in AOSD.These results indicate that, in AOSD, serum caspase-1 reflects the activation status of circulating innate immune cells.Caspase-1-dependant inflammasome activation contributes to the autoinflammatory process in AOSD, aggravating the maturation and release of proinflammatory cytokines, IL-1β and IL-18.
Our study has several limitations.First, multivariate analysis could not be performed due to the small sample size.Additionally, the analysis included a small number of patients with AOSD.Second, multiple serum cytokines were measured in patients with AOSD only, and not in control patients (RA) or HCs.Additionally, characteristic baseline differences between patients with AOSD, those with RA, and HC differ in terms of age and gender; thus, a background-aligned study is further warranted.Third, the mechanism behind caspase-1 release was not completely elucidated.Further studies require a larger number of cases and innate immune cellular analysis, which elucidates the mechanisms for elevating circulating caspase-1.

Conclusions
Serum caspase-1 levels, a component of the inflammasome, were elevated in patients with AOSD.Serum caspase-1 levels in patients with AOSD significantly correlated with disease activity indices, such as serum ferritin and Pouchet's score, as well as with inflammatory cytokines, including IL-18.Thus, caspase-1 may be an effective biomarker for diagnosing AOSD and predicting disease activity.However, the early diagnosis and prognostic value of caspase-1 in patients with AOSD require further validation with large-sample studies.

IQR:Fig 1 .
Fig 1. Serum caspase-1 levels in patients with AOSD.Serum caspase-1 levels in patients with AOSD (n = 51) were significantly higher compared to those with RA (n = 66) or healthy controls (n = 36).Comparisons of serum caspase-1 levels by the Kruskal-Wallis test are significantly different between patients with AOSD and those with RA as well as HCs.The AOSD group demonstrated significantly higher caspase-1 levels than both HCs and RA groups (p < 0.001).Post hoc pairwise analyses between three groups were analyzed by the Games-Howell test.https://doi.org/10.1371/journal.pone.0307908.g001

Fig 2 .
Fig 2. The ROC curve of caspase-1 distinguishes between AOSD and RA.The ROC curve of caspase-1 was 26.9 ng/mL for distinguishing between AOSD and RA (sensitivity of 100.0% and specificity of 93.9%).The area under the ROC curve was 0.987.https://doi.org/10.1371/journal.pone.0307908.g002 was conducted.Serum IgG fragmentation into IgG light chains (25 kDa, respectively) was detected by a secondary antibody used for western blot (Fig 6A, upper gel).Sera were pre-absorbed using protein G beads and subjected to anti-cleaved caspase-1 western blot to avoid these interfering IgG-derived bands.Singlecleaved caspase-1 bands (20 kDa) were exclusively detected in the sera from untreated patients with AOSD, not in HCs (Fig 6A, lower gel).However, cleaved caspase-1 was detected in the sera of untreated patients with AOSD, which was diminished in the paired sera after treatment (Fig 6A, lower gel).We confirmed that serum transferrin was detected, as the internal control, to the same extent in untreated and treated patients with AOSD (Fig 6B).

Fig 6 .
Fig 6.Anti-caspase-1 immunoblot analysis using sera from patients with AOSD.Sera from patients with AOSD (before or after treatments) or health HCs were dissolved in sample buffer, separated in Tris-acetate gels, and analyzed by anti-cleaved caspase-1 immunoblot analysis.(A) The cleaved caspase-1 band (p20) was detected in serum from patients with AOSD under reducing conditions.IgG light chain was detected in the upper gel, but after IgG removal by protein G beads, cleaved caspase-1 was detected as in the lower gel.(B) Serum transferrin was detected as the internal control to the same extent in untreated and treated patients with AOSD.A representative result of three independent experiments.https://doi.org/10.1371/journal.pone.0307908.g006